UC San Diego

Protein ubiquitination is a highly conserved posttranslational modification in which the protein ubiquitin is covalently linked through an isopeptide bond to a lysine residue of the target protein. Proteins can be mono-, multimono-, or poly-ubiquitinated owing to ubiquitin's seven lysine residues and N-terminal methionine, which create unique topologies and codes that can be interpreted by cellular ubiquitin receptors. Among these modifications, monoubiquitination is the most abundant and participates in a myriad of cellular processes, including protein degradation, trafficking, DNA repair, viral budding, and neurodegenerative proteinopathies. Here we demonstrate an avidity-based method that we developed, which can produce monoubiquitinated recombinant proteins with native isopeptide bonds in yields and purities sufficient for biophysical characterization. As a proof of concept, we used this method to monoubiquitinate two specific proteins, Parkinson’s protein α-synuclein and ESCRT-protein ALIX, using native NEDD4-family E3 ligases. Using quantitative chemical proteomics, we identified ubiquitination hotspots for NEDD4L-mediated monoubiquitination of α-synuclein and NEDDL, as well as WWP2-mediated monoubiquitination of ALIX. Additionally, we uncovered strikingly opposite effects of monoubiquitination on the phase separation and fibrillization properties of these two amyloidogenic proteins, thereby providing unique insights into the impact of monoubiquitination on protein aggregation.